Antimicrobial resistance (AMR) poses a critical public health challenge, particularly in low- and middle-income countries such as Vietnam. Assessing healthcare professionals' knowledge, attitudes, and practices (KAP) is vital to track progress and optimize national AMR interventions. This study aimed to assess KAP toward AMR and identify influencing factors among healthcare professionals, generating baseline evidence to strengthen stewardship and guide the implementation of Vietnam's 2023-2030 Action Plan. A cross-sectional study was conducted in six cities and provinces in Vietnam during 2023-2024, involving 3051 healthcare professionals across health system levels. Data were analyzed using multilevel linear regression, structural equation modeling (SEM), and multigroup SEM (MG-SEM) to examine KAP patterns and associated determinants. Respondents demonstrated moderate knowledge and attitude about AMR, but reported suboptimal practices. While 75.7% totally agreed that self-medication without prescription is a key cause of AMR and 66.4% rated AMR as an important community issue, only 34.2% frequently participated in AMR-related training. Moreover, only 41.1% were fully confident in educating patients on antimicrobial use. Regression analyses identified physician status, higher education, and working in provincial hospitals as key factors associated with better KAP outcomes. Notably, SEM revealed a negative association between knowledge of AMR causes and practice, and MG-SEM highlighted significant provincial variations in KAP relationships. While Vietnamese healthcare professionals show foundational awareness and support for AMR control, gaps in confidence and practice remain. These findings underscore the need for context-specific training and decentralized stewardship strategies tailored to professional roles and regional healthcare contexts.
To investigate antibiotic resistance genes (ARGs), conjugative plasmids, and virulence genes in Swedish waterborne Escherichia spp., water samples were collected from urban freshwater and Baltic Sea beaches and a primary wastewater treatment plant (WWTP) in Stockholm, Sweden. During the summer of 2022, 68 isolates were recovered using ESBL-selective and non-selective agar, including 40 from wastewater and 28 from fresh or brackish water. Isolates were characterised by phenotypic antibiotic susceptibility testing, conjugation assays, and whole-genome sequencing. Antibiotic residues were quantified, with higher concentrations detected at WWTP inlets and outlets than in natural water sources. Overall, 28 isolates (41.17%) were phenotypically multidrug-resistant (MDR), and 18 (26.47%) carried ≥3 ARGs. WWTP-derived isolates showed a significantly higher prevalence of extended-spectrum β-lactamase (ESBL) genes than freshwater isolates (p < 0.0001). Isolates represented diverse multilocus sequence types (MLST), and most harboured ≥1 plasmid. Sixteen strains transferred conjugative plasmids encoding resistance to cefotaxime, tetracycline, streptomycin, and trimethoprim. ESBL genes, including blaCTX-M-15, were located on IncF, IncN, IncB/O/K/Z, and IncI plasmids, with IncF plasmids showing lower transfer frequencies than IncN and IncI1 conjugative plasmids. These findings identify WWTPs as a major source of MDR and pathogenic E. coli, highlighting their role in environmental dissemination.
Antimicrobial resistance (AMR) is a major global health concern, involving complex transmission pathways linking humans, domestic animals, wildlife, and the environment. Wild animals and plants can harbour and transmit AMR, while also serving as sentinels. Here, we aimed to evaluate current knowledge and research gaps on AMR in wild animals and plants to inform One Health research and policy. We conducted a semi‑systematic review of AMR in wild animals and plants, generating a dataset of 866 publications and analysing metadata on host taxa, microbial and genetic targets, and analytical approaches. The literature shows strong taxonomic, geographic, and methodological biases, with mammals and birds dominating, whereas plants (n = 14) and amphibians (n = 10) were rarely studied. Resistant fungi were also under‑represented (2% of studies), while Escherichia spp. accounted for 33% of microbial targets. Employing wildlife‑based surveillance offers a useful policy tool to address key AMR gaps at human-animal-environment interfaces.
Fungal antimicrobial resistance (fAMR) is accelerating, driven in part by the dual-use of antifungal modes of action in agriculture and medicine, threatening therapy. Addressing this challenge requires a unified One Health response that balances agricultural productivity, economic stability, and human and animal health. By focusing on the United Kingdom's policy approach, we argue that current efforts are constrained by fragmented governance, surveillance and regulation. To resolve this, we propose three policy recommendations: 1. a cross-government fAMR body, 2. mandatory environmental and clinical surveillance, and 3. for fungicide approvals to look beyond crop pathogens and integrate risk assessments for potential hotspots of resistance selection in human fungal pathogens. These measures will safeguard current and future antifungals while providing much-needed regulatory clarity and will be translatable to other national and regional contexts.
Understanding all sources of selective pressure that contribute to the emergence of antibiotic resistance is essential for developing sustainable antimicrobial strategies. Here, we investigated the interaction between phage T4 and Escherichia coli MG1655 to determine whether mutations conferring phage resistance also shape the genetic background for β-lactam resistance. Using experimental evolution, whole-genome sequencing, and targeted genetic reconstructions, we identified mutations in porins and lipopolysaccharide (LPS) biosynthesis as the predominant routes to phage T4 resistance. Precise allelic replacements and isogenic strain comparisons demonstrated that these mutations not only protect against phage predation but also create a genetic context that facilitates the emergence of β-lactam resistance, including resistance to carbapenems. Together, these findings provide compelling evidence that phage-driven selection can establish bacterial genetic backgrounds predisposed to antibiotic resistance. This work highlights the evolutionary risks associated with phage therapy and underscores the need to account for genetic trade-offs when developing alternative antimicrobial strategies.
The identification of the determinants driving antimicrobial resistance is a prerequisite for improving the control of resistance emergence and dissemination. Disinfectant biocides, daily used in food-processing industries, have already been associated with the cross-selection of antibiotic-resistant bacterial populations. However, very few studies have addressed this issue using a biofilm model, the predominant bacterial lifestyle in food-processing environments. In this work, we examined the adaptation of Escherichia coli biofilms to four biocidal active substances over one month, and assessed their subsequent effects on antibiotic resistance. Exposure to N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (TMN) and benzalkonium chloride significantly increased the emergence of rifampicin-resistant (RifR) variants in biofilms. Genomic analyses revealed that the RifR variants selected upon TMN exposure recurrently harboured mutations in genes related to lipopolysaccharide (LPS) biosynthesis that conferred low-level rifampicin resistance in biofilm. These variants displayed altered LPS profiles, a more negative surface charge, and reduced membrane permeability. Proteomic and phenotypic analyses supported a metabolic reorientation of envelope sugar precursors, with decreased modulation of LPS synthesis and a marked induction of the colanic acid biosynthetic pathway in TMN-selected variants. This shift resulted in increased matrix production and reinforced biofilm-associated tolerance. Together, these data identify outer membrane reprogramming, linking LPS modulation with colanic acid overproduction, as a previously unknown mechanism of TMN adaptation that simultaneously promotes antibiotic cross-resistance in E. coli biofilms.
Black soldier fly larvae (BSFL) are promising for converting animal manure into protein; however, the risk of antibiotic resistance gene (ARG) enrichment in the larval gut during this process remains unclear. Here, we employed metagenomic and metatranscriptomic analyses to investigate this risk during BSFL conversion of duck manure. Our results demonstrated that within the BSFL treatment system, ARG abundance and diversity in manure decreased significantly over time. Concurrently, total abundance and transcriptional activity of ARGs in the larval gut were significantly lower than those in manure. However, comparative sequence analysis suggested the potential for ARG exchange between bacterial communities in manure and larval gut. Klebsiella, Escherichia, Citrobacter, and Pseudomonas were identified as the primary hosts in the gut. The enrichment and dynamics of these manure-derived ARGs were jointly driven by shifts in physicochemical properties (notably organic matter and total nitrogen), mobile genetic elements, and the bacterial community. Validation experiments demonstrated that modulating these key physicochemical drivers can mitigate ARG abundance in the larval gut. Overall, this study highlights the potential enrichment risk of manure-derived ARGs in the BSFL gut, identifies key hosts and drivers, and provides actionable mitigation strategies for safer BSFL application.
The global spread of New Delhi metallo-β-lactamase (NDM)-producing Gram-negative bacteria poses a major threat to healthcare systems worldwide, yet the processes driving their long-term establishment at national scales remain poorly understood. Here, we integrate a decade of nationwide surveillance with high-resolution genomics to reconstruct the emergence, dissemination, and evolution of blaNDM in Costa Rica, from its first detection in 2014 to a major hospital outbreak during the COVID-19 pandemic. National surveillance confirmed hundreds of blaNDM-positive isolates, revealed widespread dissemination across hospitals, and identified a temporal shift in dominant blaNDM hosts from 2020 onwards. During the pandemic, increased NDM detection coincided with a large intrahospital outbreak involving 247 patients with a 51% case-fatality rate. Genomic analysis of 40 representative isolates revealed both plasmid- and chromosome-associated blaNDM-1-carriage and heterogeneous dissemination through globally disseminated high-risk and locally emerging clones. Plasmid-resolved analyses showed that the dominant IncA/C2 blaNDM-1-carrying plasmid derived from a globally conserved backbone and diversified into multiple circulating variants and fusion events with IncF elements, generating multidrug-resistant megaplasmids. Together, these findings highlight how global connectivity, local population dynamics, and plasmid plasticity interact to drive the national persistence of blaNDM and underscore the importance of sustained genomic surveillance.
Rising antibiotic resistance causes >700,000 deaths annually and could reach ~10 million by 2050. Because the development of new antibiotics lags behind resistance, strategies that restore the activity of approved drugs are urgently needed. Gentamicin efficacy against Staphylococcus aureus and Enterococcus spp. is limited by widespread aminoglycoside-modifying enzymes (AMEs). Here, we show that palmitoleic acid, a host-derived monounsaturated fatty acid, re-sensitizes resistant gram-positive pathogens to gentamicin by accelerating intracellular drug influx rather than inhibiting AMEs. Single-cell flow cytometry and high-resolution mass spectrometry demonstrate rapid gentamicin accumulation that outpaces enzymatic inactivation. The combination lowers MICs across clinical isolates and significantly enhances killing of mature biofilms at clinically relevant concentrations. These findings establish a framework in which resistance reflects the balance between antibiotic entry and enzymatic inactivation. By shifting this balance toward drug influx, resistance can be functionally reversed without targeting resistance genes, supporting permeability-based adjuvant strategies to expand the utility of existing antibiotics.
Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a high-priority pathogen that severely limits clinical treatment options. Sewage systems serve as critical interfaces for the dissemination of antimicrobial resistance (AMR). However, the clonal distribution and evolutionary dynamics of CRKP co-harboring carbapenemases and tigecycline resistance genes in these environments remain insufficiently characterized. This study investigated CRKP resistance mechanisms in sewage using whole-genome sequencing. A total of 143 CRKP strains were isolated from 30 sewage samples (hospital, community, and livestock farm). Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) identified 22 representative strains belonging to 6 sequence types (STs), with ST11 being the predominant clone across all sources. All isolates exhibited carbapenem resistance, and 27.3% carried tigecycline resistance. The carbapenemase genes blaKPC-2 and blaNDM-1 exhibited ST-specific genetic environments. Complete genome sequencing of ST34 strain JD_E1, which carried tmexCD-toprJ, blaNDM-1, and blaKPC-2, revealed that these genes were located on conjugative IncHI5-like/IncU and IncFIB(pQil)/IncFII(Yp) plasmids. These plasmids demonstrated high transferability and stability without antibiotic selection. Plasmid pE1_NDM underwent insertion sequence (IS)-mediated structural streamlining via the deletion of non-essential segments while preserving core resistance modules. These findings suggest that sewage environments are reservoirs for multidrug-resistant CRKP clones, providing molecular epidemiological evidence for integrated infection control strategies.
The use of antimicrobials in livestock farming drives selection and dissemination of antimicrobial resistance (AMR), prompting implementation of veterinary stewardship programs to reduce antimicrobial usage (AMU). We evaluated changes in AMR on 45 Dutch pig farms before and after tailored, coaching-based interventions using phenotypic testing of Escherichia coli and metagenomic profiling of pooled faeces. Post-weaning pig farms, including nursery and fattening units, entered the intervention in a stepped-wedge design, with intervention periods ranging from 10 to 27 months. Across farms, AMU and abundances of several antimicrobial resistance gene classes declined over time, alongside reductions in overall resistome levels. Proportions of phenotypic AMR in E. coli were more variable, although decreased AMU was associated with lower resistance for specific antimicrobial classes, such as tetracyclines and beta-lactams. While longer follow-up is required to fully assess long-term impacts, these findings indicate that veterinary antimicrobial stewardship programs can yield measurable short-term reductions in AMR at farm level.
River biofilms are frequently exposed to invasion by antibiotic-resistant bacteria (ARB) due to episodic or chronic wastewater inputs, yet the ecological processes governing the fate of invaders and their resistance plasmids remain poorly understood. We experimentally exposed river-grown biofilms from sites differing in microbial diversity and wastewater impact to a genetically tagged ARB Escherichia coli carrying a transferable IncPα plasmid with the nptII resistance gene. Over two weeks, we tracked invader and plasmid dynamics using qPCR and plasmid-to-genome ratios as a proxy for horizontal gene transfer (HGT), complemented by 16S rRNA gene sequencing and metagenomics. Both quantification approaches yielded consistent results: the invader transiently established in all biofilms, peaking within 48 h and declining to near-background levels after 14 days. Decreasing plasmid-to-genome ratios indicated limited HGT and progressive plasmid loss. Biofilms impacted by wastewater showed slower declines, suggesting greater plasmid persistence in disturbed environments and increased abundance of specific indigenous antimicrobial resistance genes of public health concern. While the overall resistome exhibited short-lived shifts, and indigenous resistomes remained largely stable. These findings demonstrate that invader-biofilm interactions are dynamic and shaped by community context, supporting the One Health framework and highlighting how environmental conditions modulate antimicrobial resistance risks in freshwater ecosystems.
Methicillin-resistant Staphylococcus aureus (MRSA) is a significant public health threat due to both extensive antimicrobial resistance and immune evasion capabilities, necessitating alternative therapeutic strategies. Disruption of bacterial metal ion homeostasis, a process already leveraged by host nutritional immunity, represents a promising therapeutic approach. The synthetic ionophore PBT2 delivers zinc (Zn) directly into the bacterial cytosol, where it can dysregulate cellular processes and restore the efficacy of conventional antibiotics. Here, we use PBT2 and Zn (PZ) to study the cellular response to metal dysbiosis in MRSA, identifying new metal-dependent molecular vulnerabilities. Integrated transcriptomics, metabolomics and molecular analyses revealed that the antibacterial and oxacillin-resensitisation action of PZ is driven by dual metal stress: intracellular Zn accumulation and manganese (Mn) depletion. PZ disrupted central carbon metabolism at multiple key nodes, impairing glycolysis, the TCA cycle and respiration, leading to NADH and ATP depletion and compromised peptidoglycan biosynthesis. PZ also altered the metal-dependent oxidative stress response, resulting in superoxide accumulation. Collectively, this work presents the dynamic interplay between bacterial metal ion homeostasis, central metabolism, and β-lactam resistance. Uncovering how PBT2 subverts the adaptive responses of MRSA to host-imposed stresses contributes to our understanding of host-pathogen interactions and offers a foundation for developing novel antimicrobials based on metal homeostasis disruption.
Antifungal tolerance, unlike resistance, allows cells to grow slowly at concentrations above the minimum inhibitory concentration. While resistance mechanisms are well characterized, the pathways underlying tolerance remain elusive. Here, we performed a genetic screen of a transcriptional factor mutant library to identify regulators of azole tolerance in Candida albicans. This screen uncovered Isw2, the catalytic subunit of the ISW2 ATP-dependent chromatin remodeling complex, as a negative regulator of azole tolerance. Integrating transcriptomics, Isw2 DNA-binding profiles, and nucleosome-occupancy analyses revealed that Isw2 maintains a repressive chromatin architecture at the CRZ1 promoter, limiting the nucleosome-depleted region and suppressing CRZ1 transcription. Isw2 also modulates fluconazole heteroresistance and amphotericin B sensitivity through Crz1. In addition, we identified the copper-responsive transcription factor Mac1 as a context-dependent regulator of azole tolerance, acting negatively under copper limitation but positively under copper-replete conditions. Together, these findings reveal unexpected roles for chromatin remodeling and copper homeostasis in antifungal tolerance.
Klebsiella pneumoniae infections in young infants are an escalating threat in low- and middle-income countries, yet robust longitudinal data integrating hospital burden, clinical outcomes, antimicrobial resistance, and genomics remains scarce. We performed an 18-year (2004-2021) prospective, multicenter genomic epidemiology study across four hospitals in Bangladesh. Among 122,353 enrolled children from whom blood and/or cerebrospinal fluid cultures were performed, 1600 (1.3%) yielded culture-confirmed Klebsiella pneumoniae species complex (KpSC) isolates. Positivity increased from 16 per 1000 cases tested in 2004 to 37 per 1000 in 2021. Hospital case-fatality rate (CFR) rose from 21.4% to 51.4% during the study, paralleling the emergence and expansion of carbapenem resistance, first detected in 2008 and reaching 81% of isolates by 2021. Neonates accounted for 80.5% of infections and experienced a CFR of 40.8%. Whole-genome sequencing of 599 representative isolates revealed four KpSC species, 145 sequence types, and 92 capsular alleles. Global high-risk clones ST11, ST16, and ST147 harbouring NDM-type carbapenemases dominated recent cases. These findings document the increasing resistance and mortality associated with KpSC infections amongst neonates in Bangladesh, underscoring the urgent need for strengthened infection prevention and control, equitable access to effective combination therapies, and vaccine-based preventative strategies.
Tuberculosis (TB), the deadliest infectious disease globally, still poses an enormous public health challenge exacerbated by the rise of multi-drug resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis strains. The bicyclic nitroimidazoles pretomanid (PTM) and delamanid (DLM) represent the most recent class of anti-tubercular compounds to achieve regulatory approval and clinical implementation in TB chemotherapy regimens. Both are prodrugs whose activity relies on the deazaflavin-dependent nitroreductase Ddn. High-throughput screening on a clinically relevant DdnL49P mutant reveals molecules capable of restoring PTM activity. Optimisation through rational medicinal chemistry leads to highly potent compounds capable of drastically reducing the MIC of PTM while improving its bactericidal activity. Mechanistic studies using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), transposon sequencing and thin-layer chromatography of radiolabeled extracted lipids show that these molecules do not trigger bioactivation pathways, but instead target a compensatory pathway involving Rv2073c, an enzyme that is redundant with DprE2 for bacterial cell wall biosynthesis. The concomitant perturbation of both the DprE2- and Rv2073c-dependent steps by the PTM-NAD adduct and optimised norbornene derivatives leads to a collapse in arabinan synthesis correlating with bacterial death. This study thus highlights Rv2073c as a promising vulnerability that can be exploited to potentiate the efficacy of nitroimidazole anti-tuberculosis drugs.
There is a rising concern that temperature increases associated with climate change might select for antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquatic ecosystems, although the extent of this phenomenon remains unknown. This study aimed to determine whether increased temperatures induce ARG selection and modify the transcriptional response in a model aquatic ecosystem using an omics approach. River water microcosms were incubated at 20 °C, at 28 °C (constant temperature increase), and under oscillating temperatures (intervals of 3 days at 20 °C and 4 days at 28 °C to emulate heatwaves) for 28 days. Both the constantly higher temperature and oscillating temperatures altered bacterial community composition and selected for members carrying ARGs, including two Limnohabitans subpopulations that contain the pmrE polymyxin resistance gene and an Alphaproteobacteria carrying the sul2 sulfonamide resistance gene. In addition, metatranscriptomic analyses revealed a lower abundance (p < 0.05) of transcripts related to cell division, bacterial activity/metabolism, antibiotic efflux, stress responses, and cell/gene mobility in samples exposed to oscillating temperatures than in the room temperature controls. This research supports the rising concern that climate change may increase antibiotic resistance in environments exposed to higher temperatures maintained over long periods of time and to repeated short-time heatwave events.
Escherichia coli is a major cause of deadly infections in calves. As surveillance in livestock is mainly performed on carriage isolates, factors driving the dissemination of E. coli infecting calves remain largely unknown. Here, we analyzed the population of β-lactam-resistant E. coli in sick calves across five calving seasons on 444 farms in Wallonia, Belgium. 99% of the isolates were MDR and enriched in virulence factors, adhesins and toxins. Restrictions on critical antibiotic usage have no impact on population structure and β-lactamase gene content. This resilient population is likely shaped by virulence and resistance. Correlations of phylogeny and geographic origin suggest indirect local transmission with differences between regions east and west of the Meuse. Phylogenetic analyses with isolates from EnteroBase show a close relationship of calf isolates with human isolates, suggesting host jumps. These findings provide new means to further model the spread of E. coli in livestock farming.
Antimicrobial resistance (AMR) poses a growing global threat, with low- and middle-income countries (LMICs) bearing a disproportionate burden of resistant infections. Detection of resistance is critical for guiding treatment, enabling stewardship, and supporting surveillance, yet diagnostic capacity in LMICs remains severely limited. We conducted a scoping review to map low-cost diagnostic approaches suitable for resource-limited settings for detecting AMR in bacterial infections, excluding tuberculosis. Following PRISMA-ScR guidelines, we screened 585 articles and included 61 studies, which we evaluated using a framework based on the WHO REASSURED criteria. Genotypic approaches predominated (39 studies) while 22 studies employed phenotypic approaches. The majority (72%) were proof-of-concept studies, only 8 were implemented in LMICs, and just 2 assessed clinical outcomes. Critical gaps included limited validation on clinical specimens, near-absent cost data, high infrastructural requirements, and inconsistent reporting of performance metrics. To address reporting heterogeneity and improve knowledge gaps, we propose the REACH checklist (Readiness and design, Execution and workflow, Access requirements, Clinical and analytical performance, and Health outcomes and economics) as a reporting framework derived directly from the gaps identified across included studies, intended to span AMR diagnostic development from proof-of-concept through clinical validation relevant to low-resource settings.
Aquatic environments are key reservoirs and dissemination pathways of antimicrobial resistance (AMR). However, current water-based surveillance remains fragmented and inefficient for the timely detection of emerging threats. Integrating artificial intelligence with embedded metadata provides a powerful pathway to identify novel antimicrobial resistance genes, characterize resistome profiles, and predict AMR dynamics in real-time by combining omics, environmental, and hydrological data into spatiotemporal predictive models. Successful implementation of this framework will require robust governance, ethical safeguards, and capacity building to support predictive AMR monitoring aligned with the One Health approach.